The isolation of halophilic urease-producing bacteria and the study of their nano-crystal production

Urease-producing bacteria can precipitate calcite nano-crystals by producing urease in the presence of urea and calcium. Calcite precipitation resulting from microbial activity is a process which causes cementation of soil particles in nature. The purpose of this study was to isolate urease-producing halophilic bacteria in order to precipitate calcite in saline soil. Natural samples, including soil and saline waters, were selected for this purpose. At First, halophilic bacteria were isolated by salt-containing TSB medium. Then, a selective medium containing phenol red and urea facilitated the isolation of urease-producing bacteria. Hydrolysis of urea by urease causes alkalization of the medium and the formation of pink halo around colonies. Finally, the best isolate was selected for further study by measuring the release of ammonium by the Nessler method. The ability or inability of isolates to produce calcite was investigated by culturing the isolates on sedimentary medium with different salt concentrations for 10 days. In total, 110 halophilic isolates were isolated, among which 58 isolates had the ability of urease production. The microscopic studies of colonies showed that only 6 isolates were able to produce crystals on precipitation medium. Isolate 6 was selected for further study and then analyzed by X-ray diffraction crystals on precipitation medium. Isolate 6 was selected for further study and then analyzed by X-ray diffraction (XRD) and scanning electron microscope (SEM) equipped with an energy dispersive X-ray (EDX) detector. Studies of urease activity showed that this strain released 20.86 mM ammonium after 18 hours. This bacterium was identified by biochemical and molecular analyses and the comparison of its 16S rRNA gene sequence showed 99.92% similarity with the similar gene sequence in Staphylococcus xylosus and then this sequence was submitted in NCBI database with the accession number MG655155. This isolate was able to produce calcite in free salt medium, with salinity up to 10%. Nowadays, many efforts have been made to produce environmental-friendly cements, and therefore, the use of urease-producing halophilic bacteria is an appropriate candidate for bio-cementing in saline environments.